| Glutamine(Gln)is a kind of non-essential amino acid in animals,which plays a crucial role in enhancing the immunity.However,Gln has low solubility and is prone to decomposition into pyroglutamic acid and ammonia when heated,which can be toxic to animals.To overcome these limitations,Alanyl-glutamine(Ala-Gln)is synthesized from alanine and glutamine.Ala-Gln not only circumvents the drawbacks of Gln but also undergoes hydrolysis into Gln in vivo,facilitating absorption by the intestinal mucosa.This enables Ala-Gln to effectively fulfill the role of Gln.In this study,the researchers investigated the effects of Ala-Gln on the immune function of Paramisgurnus dabryanus,assessed its alleviating effect on intestinal inflammation induced by Aeromonas hydrophila,and elucidated the underlying mechanism of action.The main research contents and results are summarized as follows:In Experiment 1,Paramisgurnus dabryanus with initial body weight of(4.07 ±0.01)g were fed a basic diet supplemented with different levels of Ala-Gln: 0.00%(control group),0.25%,0.75% and 1.25% respectively,for a duration of 70 days.Various immune and antioxidant parameters of serum,liver and intestinal were measured,intestinal morphology was observed.The results revealed that at a dietary supplemental level of 0.75% Ala-Gln,the levels of AKP,LZM,C3 and C4 in serum,as well as C3 and C4 in the intestine,were significantly higher compared to the control group(P < 0.05).Ala-Gln had a significant impact on the activities of CAT,SOD,TAOC,and MDA in the liver(P < 0.05).When the supplemental level was increased to1.25%,the levels of CAT,T-AOC,and SOD in the liver were significantly higher compared to the 0.00% group(P < 0.05).With increasing dietary Ala-Gln content,the MDA content in the liver initially decreased significantly and then increased(P < 0.05),reaching the lowest level in the 0.75% group.The histological examination of intestinal tissue slices indicated that dietary supplementation with Ala-Gln promoted the development of intestinal villi,resulting in increased villi height and muscle thickness.In Experiment 2,Paramisgurnus dabryanus were divided into five groups with three replicates per group and five Paramisgurnus dabryanus per replicate.Four groups were designated as experimental groups,and each Paramisgurnus dabryanus was intraperitoneally injected with 100 μL of Aeromonas hydrophila at concentrations of1.5×109,1.5×108,1.5×107 and 1.5×106 CFU/m L,respectively.The remaining group served as the control group and received an intraperitoneal injection of 100 μL of PBS.Within 24 hours after injection,the mortality of Paramisgurnus dabryanus in each group was recorded,and the mortality rate and semi-lethal concentration were calculated.The results revealed that the semi-lethal concentration of Aeromonas hydrophila for Paramisgurnus dabryanus was determined to be 1.17×109 CFU/m L.In Experiment 3,the control group of Paramisgurnus dabryanus was intraperitoneally injected with Aeromonas hydrophila after 70 days of culture in Experiment 1.Four Paramisgurnus dabryanus were sampled at 0 hour before infection and 6 hours,12 hours and 24 hours after infection.Blood samples were collected,and intestinal tissues were obtained for transcriptome sequencing analysis.The results of the analysis revealed significant enrichment of immune-related pathways,such as the pattern recognition receptor signaling pathway and the complement and coagulation cascade,among the differentially expressed genes identified using the KEGG pathway enrichment analysis.Furthermore,several differentially expressed genes associated with immunity,including TLR5,My D88,TNF-α,C3 and CD59 were identified.These findings elucidated the pathogenic mechanism of Aeromonas hydrophila infection in Paramisgurnus dabryanus.According to the results of Experiment 1,2 and 3,Experiment 4 was conducted to investigate the impact of different levels of Ala-Gln supplementation on Paramisgurnus dabryanus during Aeromonas hydrophila infection.The immune and antioxidant parameters of the intestine and liver were measured,and tissue sections were observed at 0 hour(pre-infection),6 hours,12 hours,and 24 hours post-infection.Additionally,transcriptome analysis was performed on the intestinal samples.The results demonstrated significant differences in the activities of CAT,SOD,and T-AOC in the liver of the Aeromonas hydrophila injection group at each time point(P < 0.05).These activities displayed an initial increase followed by a subsequent decrease,returning to the pre-injection level after 24 hours.Notably,the activities of CAT and SOD in the0.75% group were significantly higher than those in the other groups at all time points after infection(P < 0.05).Moreover,the activity of T-AOC in the 1.25% group was significantly higher than that in the 0.00% group throughout the infection period(P <0.05),while the 0.75% group exhibited significantly higher T-AOC activity than the0.00% group at 12 hours and 24 hours post-infection(P < 0.05).The activity of MDA in the 0.75% and 1.25% groups exhibited significant changes with the duration of infection,peaking at 12 hours and subsequently decreasing significantly at 24 hours(P< 0.05).As the infection time increased,the activities of AKP,LZM,C3,and C4 in the intestine showed significant fluctuations(P < 0.05),reaching their lowest values at 12 hours.Notably,the activity of AKP in the 0.75% group was significantly higher than that in the 0.00% group at all time points after infection(P < 0.05).Conversely,the activity of LZM in the 0.00% group at 12 hours was significantly higher than that in the0.00% group(P < 0.05),and the activities of C3 and C4 in the 0.75% group at 12 hours and 24 hours were significantly higher than those in the other groups(P < 0.05).Observations of liver and intestinal tissue sections revealed tissue damage in all groups following Aeromonas hydrophila infection,with noticeable improvements observed 24 hours later.The liver and intestinal tissue structures of the 0.75% and 1.25% groups appeared more intact compared to the other groups.Transcriptome analysis of the intestinal tracts in each group revealed the largest number of differentially expressed genes(DEGs)enriched in most pathways at 12 hours post-infection,exhibiting a pattern of initial increase followed by a decrease over the course of infection(6 hours to 24hours).At 0 hour prior to infection,significant differences in immune diseases were observed between the Ala-Gln treatment group and the control group.At 6 hours and12 hours post-infection,the metabolic and immune-related differences between the treatment groups and the control group increased significantly.By 24 hours postinfection,the immunity of the treatment groups began to show some signs of recovery compared to the control group,with the 0.75% Ala-Gln dosage exhibiting better effects.Furthermore,Ala-Gln was found to alleviate intestinal inflammation caused by Aeromonas hydrophila infection through the regulation of MHC,CTSB,TNF-α,and C3 expression within the pathways of antigen processing and presentation,complement coagulation and cascade.In conclusion,the supplementation of diets with 0.75% to 1.25% Ala-Gln has been found to enhance intestinal immunity and mitigate intestinal injury in Paramisgurnus dabryanus.These findings suggest that Ala-Gln supplementation may potentially alleviate intestinal inflammation in Paramisgurnus dabryanus by modulating the pathways involved in antigen processing and presentation,complement coagulation,and cascade. |
